Our research is investigating chromatin structure and function as revealed by single molecule approaches. Single molecule approaches (e.g. atomic force microscopy AFM, optical tweezers) can answer particular questions that are difficult (if not impossible) to answer by population-ensemble experiments (e.g. gel electrophoresis etc.). Our interests in chromatin are (i) linker and core histones (variants, stoichiometry) and their contributions to fiber structure, (ii) post-translational modifications of histones (acetylation, phosphorylation, ADP-ribosylation, etc.) affecting structure, (iii) effect of DNA methylation on fiber structure, and (iv) interactions of chromatin fibers or single nucleosomes reconstituted on specific DNA sequences with other macromolecular complexes involved in DNA functioning (e.g. polymerases, chromatin remodeling factors, etc.). Subproject 1: AFM imaging and manipulations: With AFM our approach is twofold: on one side, high-resolution imaging of protein/DNA complexes (chromatin fibers of various composition, stoichiometry and post-translational modifications), and, on the other side, direct manipulation of single chromatin fibers with the AFM tip to probe the forces holding the structure of the chromatin fiber together. Force is a factor in many processes involving chromatin and chromosome structural reorganizations during the life of any eukaryotic cell. Force may be needed to clear histones from the DNA for biological processes such as transcription, replication and repair. Force generation and application to biological structure is a major component of dynamic biological processes, but forces governing chromatin structure and function have not been experimentally approached up to now. Our studies of pulling short reconstituted chromatin fibers nonspecifically attached to the tip and surface lead us to conclude that we were measuring the force of adhesion of the nucleosomes to the glass substrate. To circumvent this experimental artifact, we are now tethering the DNA ends between the AFM tip and the surface, with the idea of directly reconstituting chromatin onto a single DNA molecule (similar to the optical tweezers experiments, see below). Subproject 2: Force measurements with optical tweezers Our interests in the effect of force applied to chromatin fibers has lead us to collaborate with researchers at the University of Twente (The Netherlands) on applying optical tweezers to chromatin fibers. With the optical tweezers we can probe the lower region of forces 1-150 picoNewtons (pN), whereas with the AFM we can probe forces above 100 pN. In these optical tweezers experiments, we first attached a piece of DNA between two beads, demonstrated that it was an intact single molecule of DNA that can undergo the well known B-DNA to S-DNA transition, and then assembled histones onto this single DNA molecule by injecting a Xenopus laevis egg nucleosome assembly extract into the liquid cell of the instrument. These kinds of experiments open a whole new approach because with the nuclear extract it is possible to assemble chromatin with various complements of histones (i.e., only core histone H3/H4 tetramers, fluorescently modified histones, with or without linker histone subtypes, etc.). We have found that our optical tweezers set-up is sensitive enough to detect the disruption of single nucleosomes among the ~240 nucleosomes assembled on the 48,502 bp of lambda DNA. We have determined that a range of forces of 15 pN to 40 pN is sufficient to unravel a single nucleosome. The unraveling of an individual nucleosome results in a length increase of the chromatin fiber of ~65 nm. This length increase is on the order of the length of two wraps of DNA around the nucleosome core particle. We believe that our measurements are fundamental to understanding the dynamic changes in nucleosome structure as nucleosomes are formed and then disassembled to allow access of transcription, replication and repair machineries to the underlying DNA template. Subproject 3: DNA methylation and chromatin structure Methylation of certain bases is the sole post-synthetic modification in DNA. Methylation of cytosine takes place in CpG dinucleotides. Concentrations of methylatable CpGs form the so-called CpG islands; the methylation status of CpG islands in enhancers/promoters of genes determine the transcriptional activity of the gene (CpG islands downstream of initiation sites do not affect transcription). Since methylation of CpG islands in promoters blocks transcription, we have been investigating a possible methylation-dependent chromatin compaction. We are using both AFM imaging and biochemical approaches to study this issue. We found that chromatin fibers hypermethylated in vivo were more compact than fibers isolated from control fibers. Modeling studies suggest that more DNA is wrapped around nucleosomal particles in the methylated chromatin fibers than in the control fibers. In vitro studies point to a cooperation between CpG methylation and linker histone binding in the formation of more compacted fibers; DNA methylation or linker histone binding alone do not cause fiber compaction. Subproject 4: Archael protein HMf (Histone from Methanothermus fervidus) has the same histone-fold structure as the eukaryal core histones although it lacks completely the post-translationally modified histone tails. We have investigated the ability of this protein to form chromatin fiber structure to gain insights into possible similarities and differences between eukaryal chromatin and its Archael counterpart. We have found that this protein can indeed be reconstituted onto DNA to form bona fide chromatin fibers as revealed by AFM and biochemical studies. Interestingly, the lessor stability of Archael mononucleosomes and short oligonucleosomes suggests that the post-translationally modifiable tails of the eukaryal histones lead to greater stability of eukaryal chromatin as well as serve to regulate accessibility to the underlying DNA template for DNA functioning. Subproject 5: Magnetic tweezers manipulation of single chromatin fibers We have developed a magnetic tweezers instrument to analyze single chromatin fibers in real time. In preliminary results, we have been able to manipulate a single DNA molecule and also to assemble the single DNA into chromatin using a purified system of nucleosome assembly protein 1 and core histones. With this system we should be able to study the effects of external force and torsion on a single chromatin fiber, and should get data that rivals the sensitivity of our recently published optical tweezers data. Collaborators in alphabetical order: Martin Bennink, Ph.D., Univ. of Twente, Enschede, The Netherlands David Brown, Ph.D., University of Mississippi Medical Center, Jackson, Mississippi Paola Caiafa, Ph.D., Univ. "La Sapienza", Rome, Italy Paul Smith, ODS Jordanka Zlatanova, Ph.D., Dr.Sc., Polytechnic University, Brooklyn, New York